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Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins

Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes...

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Autores principales: Hiatt, Joseph, Cavero, Devin A., McGregor, Michael J., Zheng, Weihao, Budzik, Jonathan M., Roth, Theodore L., Haas, Kelsey M., Wu, David, Rathore, Ujjwal, Meyer-Franke, Anke, Bouzidi, Mohamed S., Shifrut, Eric, Lee, Youjin, Kumar, Vigneshwari Easwar, Dang, Eric V., Gordon, David E., Wojcechowskyj, Jason A., Hultquist, Judd F., Fontaine, Krystal A., Pillai, Satish K., Cox, Jeffery S., Ernst, Joel D., Krogan, Nevan J., Marson, Alexander
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8188731/
https://www.ncbi.nlm.nih.gov/pubmed/33979618
http://dx.doi.org/10.1016/j.celrep.2021.109105
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author Hiatt, Joseph
Cavero, Devin A.
McGregor, Michael J.
Zheng, Weihao
Budzik, Jonathan M.
Roth, Theodore L.
Haas, Kelsey M.
Wu, David
Rathore, Ujjwal
Meyer-Franke, Anke
Bouzidi, Mohamed S.
Shifrut, Eric
Lee, Youjin
Kumar, Vigneshwari Easwar
Dang, Eric V.
Gordon, David E.
Wojcechowskyj, Jason A.
Hultquist, Judd F.
Fontaine, Krystal A.
Pillai, Satish K.
Cox, Jeffery S.
Ernst, Joel D.
Krogan, Nevan J.
Marson, Alexander
author_facet Hiatt, Joseph
Cavero, Devin A.
McGregor, Michael J.
Zheng, Weihao
Budzik, Jonathan M.
Roth, Theodore L.
Haas, Kelsey M.
Wu, David
Rathore, Ujjwal
Meyer-Franke, Anke
Bouzidi, Mohamed S.
Shifrut, Eric
Lee, Youjin
Kumar, Vigneshwari Easwar
Dang, Eric V.
Gordon, David E.
Wojcechowskyj, Jason A.
Hultquist, Judd F.
Fontaine, Krystal A.
Pillai, Satish K.
Cox, Jeffery S.
Ernst, Joel D.
Krogan, Nevan J.
Marson, Alexander
author_sort Hiatt, Joseph
collection PubMed
description Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14(+) human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically edited cells that retain transcript and protein markers of myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection >50-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible, and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate the development of myeloid cellular therapies.
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spelling pubmed-81887312021-06-09 Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins Hiatt, Joseph Cavero, Devin A. McGregor, Michael J. Zheng, Weihao Budzik, Jonathan M. Roth, Theodore L. Haas, Kelsey M. Wu, David Rathore, Ujjwal Meyer-Franke, Anke Bouzidi, Mohamed S. Shifrut, Eric Lee, Youjin Kumar, Vigneshwari Easwar Dang, Eric V. Gordon, David E. Wojcechowskyj, Jason A. Hultquist, Judd F. Fontaine, Krystal A. Pillai, Satish K. Cox, Jeffery S. Ernst, Joel D. Krogan, Nevan J. Marson, Alexander Cell Rep Article Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14(+) human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically edited cells that retain transcript and protein markers of myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection >50-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible, and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate the development of myeloid cellular therapies. 2021-05-11 /pmc/articles/PMC8188731/ /pubmed/33979618 http://dx.doi.org/10.1016/j.celrep.2021.109105 Text en https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ).
spellingShingle Article
Hiatt, Joseph
Cavero, Devin A.
McGregor, Michael J.
Zheng, Weihao
Budzik, Jonathan M.
Roth, Theodore L.
Haas, Kelsey M.
Wu, David
Rathore, Ujjwal
Meyer-Franke, Anke
Bouzidi, Mohamed S.
Shifrut, Eric
Lee, Youjin
Kumar, Vigneshwari Easwar
Dang, Eric V.
Gordon, David E.
Wojcechowskyj, Jason A.
Hultquist, Judd F.
Fontaine, Krystal A.
Pillai, Satish K.
Cox, Jeffery S.
Ernst, Joel D.
Krogan, Nevan J.
Marson, Alexander
Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins
title Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins
title_full Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins
title_fullStr Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins
title_full_unstemmed Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins
title_short Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins
title_sort efficient generation of isogenic primary human myeloid cells using crispr-cas9 ribonucleoproteins
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8188731/
https://www.ncbi.nlm.nih.gov/pubmed/33979618
http://dx.doi.org/10.1016/j.celrep.2021.109105
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